Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health

Authors


Correspondence: Todd R. Klaenhammer, Southeast Dairy Foods Research Center, Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, 339 Schaub Hall, 400 Dan Allen Dr., Box 7624, Raleigh, NC 27695, USA. Tel.: 919 515 2972; fax: 919 513 0014; e-mail: klaenhammer@ncsu.edu

Abstract

Certain lactic acid bacteria (LAB) have the capacity to occupy mucosal niches of humans, including the oral cavity, gastrointestinal tract, and vagina. Among commensal, LAB are species of the acidophilus complex, which have proven to be a substantial reservoir for microorganisms with probiotic attributes. Specifically, Lactobacillus gasseri is an autochthonous microorganism which has been evaluated for probiotic activity based on the availability of genome sequence and species-specific adaptation to the human mucosa. Niche-related characteristics of L. gasseri contributing to indigenous colonization include tolerance of low pH environments, resistance to bile salts, and adhesion to the host epithelium. In humans, L. gasseri elicits various health benefits through its antimicrobial activity, bacteriocin production, and immunomodulation of the innate and adaptive systems. The genomic and empirical evidence supporting use of L. gasseri in probiotic applications is substantiated by clinical trial data displaying maintenance of vaginal homeostasis, mitigation of Helicobacter pylori infection, and amelioration of diarrhea.

Introduction

Probiotics are ‘live microorganisms, which when administered in adequate amounts, confer a health benefit upon the host’ (FAO/WHO, 2002). Probiotic microorganisms are associated with several health benefits related to the maintenance of mucosal homeostasis and the immune system, which have the potential for improving human health through prophylactic and therapeutic applications in the gastrointestinal tract (GIT) and vagina (Bron et al., 2011). Many of the well-characterized probiotic strains are lactic acid bacteria (LAB), which are a phylogenetically diverse clade of Gram-positive eubacteria that ferment glucose with lactate as a primary metabolic end product. LAB are primarily adapted to nutrient-rich environments, including plant and food ecosystems as well as the mammalian mucosa. They are among the first bacteria to colonize the GIT along with Bifidobacterium, although the latter is predominant in early colonization (Reuter, 2001). The oral cavity is also considered a reservoir for intestinal bacteria and is a common mucosal niche for LAB (Dal Bello & Hertel, 2006). Probiotic bacteria are predominantly of the genera Lactobacillus and Bifidobacterium, with some notable exceptions (de Vrese & Schrezenmeir, 2008). Lactobacillus gasseri is a prolific autochthonous microorganism that colonizes the GIT, oral cavity, and vagina in humans. Lactobacillus gasseri is classified as a group B acidophilus complex microorganism, and can be differentiated from group A members by the way of genetic determination and the apparent absence of major surface-layer (S-layer) proteins (Pot et al., 1993; Boot et al., 1996). The niche-related phenotypes involved in colonization of the human mucosa, including the oral cavity, GIT, and vagina are exhibited by LAB such as L. gasseri and may contribute to or potentiate probiotic activity.

Commercial probiotic cultures belonging to the acidophilus group, such as Lactobacillus acidophilus with three S-layer proteins (Goh et al., 2009), have been used as probiotics for over 40 years. Lactobacillus acidophilus NCFM is a widely consumed and well-established probiotic microorganism, for which double-blinded, randomized, placebo-controlled clinical trials have substantiated its safety and propensity to positively influence human health. Specifically, administration of L. acidophilus NCFM has been associated with amelioration of viral infection symptoms, improvement of parameters related to gut function, and decreases in the Clostridium difficile subpopulation of the microbiota (Leyer et al., 2009; Ouwehand et al., 2009; Ringel-Kulka et al., 2011; Lahtinen et al., 2012). The genomic sequence of L. acidophilus NCFM has facilitated functional analysis of key genetic determinants that contribute to its survival and activity in the human GIT, which further adds credence to its status as a probiotic (Altermann et al., 2005). Probiotic activity is contingent upon survival during GIT passage and transient domination of the small intestinal microbiota, where adhesion of probiotic microorganisms to the host epithelium facilitates differential gene expression and immunomodulation (Booijink et al., 2007; van Baarlen et al., 2011). Microorganism–host crosstalk by probiotic microorganisms is also implicated in contributing to epithelial barrier integrity by eliciting up-regulation of tight junction proteins, increased defensin production by paneth cells, and increased mucin production by goblet cells (Mack et al., 2003; Schlee et al., 2007, 2008; Anderson et al., 2010; Karczewski et al., 2010). The interaction of probiotics with resident gut microorganisms may also induce differential expression in the host microbiota, contributing to GIT homeostasis or causing direct antagonism of enteric pathogens (Corr et al., 2007; Lahtinen et al., 2012).

For the application of new probiotic species, proper identification and considerable in vitro, in vivo, and clinical evidence must justify their use in foods or supplements for safe consumption by the greater population (Salminen et al., 1998). Several criteria for the selection of probiotic microorganisms have been established by collaborations through the LAB industrial platform (LABIP), as outlined in Table 1. There is an increasing body of evidence that indicates L. gasseri has significant potential for probiotic application by fulfilling these criteria.

Table 1. Probiotic selection criteria
  1. Modified from Dunne et al. (1999)

Human origin
Nonpathogenic, nontoxigenic and noninvasive
Devoid of transmissible antibiotic resistance genes
Resistance to technological processes
Demonstrated acid and bile tolerance
Adhesion to epithelial tissue
Transient persistence in the GIT
Produce antimicrobial substances
Antagonize pathogens and prevent infection
Modulate immune responses
Positively influence metabolic activity

Important criteria for probiotic candidacy are safety and efficacy (Gasser, 1994; Salminen et al., 1998). Proper identification of probiotic microorganisms is essential because of the strain-specific nature of the health benefits associated with probiotic cultures and the intrinsic safety issues associated with erroneous identification (Sanders et al., 2010). Initially, L. gasseri was indistinguishable from L. acidophilus by phenotypic and metabolic analysis and was not classified as a separate organism until after distinct subgroups were observed in the electrophoretic characterization of lactate dehydrogenases from L. acidophilus (Gasser, 1970; Gasser et al., 1970). The difficulty in distinguishing L. acidophilus and L. gasseri by cell morphology and biochemical means necessitated the development of practical hybridization techniques that could differentiate these two species (Luchansky et al., 1991; Pot et al., 1993). Lactobacillus gasseri was defined as a separate organism based on DNA-DNA hybridization techniques (Lauer & Kandler, 1980; Lauer et al., 1980). Since then, other nucleic acid-based methods have been developed to distinguish among closely related members of the acidophilus complex, including comparison of the variable region (V1 and V2) sequences within the 16S rRNA gene and repetitive element-PCR (Rep-PCR) using primers specific for lactobacilli (Kullen et al., 2000; Gevers et al., 2001). However, the utility of 16S rRNA gene sequences in distinguishing L. gasseri from L. acidophilus and L. johnsonii is limited due to the high sequence similarity of the 16S rRNA genes between the species. Instead, the specific design of primers for generating amplicons unique to L. gasseri, enabled by whole genome sequence analysis, offers a greater distinction from other acidophilus complex microorganisms (Klaenhammer et al., 2005).

Characteristics of probiotic strains

Following proper taxonomic identification of a probiotic candidate, the strain must be evaluated for the traits listed in Table 1, for which genome sequences are indispensible. Notably, sequencing facilitates differentiation at the strain level and identification of unsavory genes constituting exclusion criteria, including intact virulence genes and transmissible antibiotic resistance genes. Putative probiotic strains should be nonpathogenic, noninvasive, nontoxigenic, and exhibit acceptable antibiotic resistance profiles. Toxicological assays and epidemiological tracking demonstrate that commercial probiotic microorganisms have a low propensity to cause septicemia or adverse side effects when consumed in large doses in healthy individuals (Donohue et al., 1993; Saxelin et al., 1996; Salminen et al., 2002). The widespread colonization of mucosal niches by L. gasseri indicates its prevalence as a commensal in healthy individuals (Fig. 1) and the absence of any association with a health detriment affirms its safety (De Backer et al., 2007; Delgado et al., 2007; Hojo et al., 2007; Hernández-Rodríguez et al., 2011). In fact, L. gasseri is commonly found in infants and is one of the predominant species involved in early colonization of the GIT and is persistent throughout adulthood (Wall et al., 2007). The colonization of the vagina by L. gasseri and its early colonization of the infant GIT seem to indicate a potential route of transfer to neonates (Brook et al., 1979; Fanaro et al., 2007). The genomic sequence of L. gasseri ATCC 33323 revealed the absence of transmissible antibiotic resistance genes and other unsavory genes, beyond niche-related genes that facilitate adherence or toxin and drug efflux systems (Azcarate-Peril et al., 2008). Despite this, it cannot be assumed that all strains of L. gasseri are devoid of transmissible antibiotic resistance genes, necessitating the evaluation of antibiotic resistance and genome sequencing of each putative probiotic strain prior to commercialization.

Figure 1.

Cultivable levels of Lactobacillus gasseri throughout the human mucosa. Colonization of mucosal niches by L. gasseri is highly variable among individuals, ranging from undetectable to 109 CFU mL−1 in the small and large intestinal tract. Although L. gasseri is also commonly isolated from breast milk, the source and level of inhabitance remain to be investigated.

Investigation of the oral toxicity of L. gasseri CECT5714 was conducted by administration to Balb/c mice at 1010 CFU day−1 for 30 days (Lara-Villoslada et al., 2007a, b). Bacterial translocation and mucosal barrier integrity were assayed, as well as serum parameters for stress. The extended treatment did not increase bacterial translocation and did not significantly change any biochemical or hematological parameters, including degradation of mucin. The strain exhibited no significant resistance to clinically relevant antibiotics. The results from the toxicological mouse assays with L. gasseri were corroborated with feeding studies in humans with the same dose, but a duration of 5 days (Hütt et al., 2011). Markers for gut homeostasis, hepatic, and kidney function were not significantly affected by the treatment, nor were blood parameters.

Despite the established safety of this microorganism in healthy humans, it is pertinent to note that probiotic microorganisms used for therapeutic purposes may be administered to individuals with compromised epithelial barrier function and immune systems, as in the elderly, neonates, and those predisposed to autoimmune diseases (Sanders et al., 2010). These individuals may be more susceptible to septicemia or adverse side effects of probiotic administration. It is of interest, however, that even in individuals with compromised barrier integrity or immune system function, therapeutic administrations of probiotic strains did not increase translocation or incidence of septicemia (Leyer et al., 2009; Sanders et al., 2010). Due to the inherent complexity of human health and the ecosystem of the GIT it is difficult to predict the safety of a particular probiotic culture, especially when intended for compromised individuals. Moreover, studies indicating clinical safety cannot be extrapolated to closely related strains or across different host systems.

Genomic basis for in vitro survival and activity

Probiotic cultures must remain viable in the delivery vehicle (food product or capsule) throughout shelf life and during transit through the GIT to effectively retain probiotic activity. Growth conditions, processing parameters, vehicle characteristics, and the stress response of probiotic microorganisms dictate viability in these disparate environments. Survival and transient colonization of probiotic microorganisms in the GIT depends on acid tolerance, bile salt resistance, adhesion capacity, metabolic capacity, and several host-dependent factors. Consequently, it is necessary to highlight the role of genomic sequencing in the selection process for probiotic microorganisms as it enables in silico evaluation of efficacy and offers pan-genome analysis of genotypes and regulatory networks involved in probiotic mechanisms. Genome-wide comparison of the acidophilus complex microorganisms indicates considerable synteny between L. acidophilus and L. johnsonii, but greater similarity exists between the group B microorganisms L. gasseri and L. johnsonii (Klaenhammer et al., 2005). The genomic sequence of L. gasseri ATCC 33323 reveals a mechanistic basis for its survival in GI passage as well as potential probiotic genotypes. However, the influence of probiotic microorganisms on human health is dependent on activity in the host; accordingly expression analysis, as well as in vitro and in vivo testing of key genotypes, is necessary to gauge the relevance of genomic features.

Acid and bile exposure during GI passage constitute significant hurdles for probiotic microorganisms to survive and retain activity in the GIT (Sanderson, 1999). LAB encode several acid tolerance mechanisms including F1F0 ATPases and ornithine decarboxylases to maintain stable intracellular pH in acidic environments. Lactobacillus gasseri ATCC 33323 encodes GroEL, GroES, DnaK, DnaJ, and Clp protease chaperones to protect against intracellular aggregation of proteins during stress (Azcarate-Peril et al., 2008). Bile salt hydrolases and bile salt transporters have been shown to confer bile tolerance to commensal microorganisms in the GIT and also represent important attributes for probiotic survival (Begley et al., 2006a, b). The genome of L. gasseri encodes an operon containing two bile salt transporters and a bile salt hydrolase with considerable homology to the corresponding proteins in L. acidophilus NCFM and L. johnsonii NCC533. Lactobacillus gasseri also contains a bile salt hydrolase homologue that specifically hydrolyzes bile salts conjugated to taurine and an operon similar to one in L. acidophilus that is demonstrated to influence bile tolerance (McAuliffe et al., 2005; Azcárate-Peril et al., 2006; Pfeiler et al., 2007). Interestingly, the in vitro bile tolerance of L. gasseri and L. acidophilus was pH-dependent, with L. gasseri demonstrating an order of magnitude higher than bile tolerance at pH 6 than L. acidophilus. Analysis of several L. gasseri strains for survival in simulated gastric juice yielded strain-specific viability, but in general L. gasseri strains were acid- and bile-tolerant (Fernández et al., 2003; Strahinic et al., 2007; Azcarate-Peril et al., 2008; Jensen et al., 2012).

Adhesion and possible penetration of the mucus layer are important probiotic attributes that may contribute to transient colonization of the GIT and competitive exclusion of pathogens. The capacity to penetrate the mucus layer may also promote direct interaction with intestinal epithelial cells and resident immune system cells of the lamina propria (Sánchez et al., 2008). Adhesion to mucus, the extracellular matrix, and intestinal epithelial cells is mediated by several factors on the cell surface of Gram-positive microorganisms, including lipoteichoic acid, mucus-binding proteins, fibronectin-binding proteins and S-layer proteins (Granato et al., 1999; Buck et al., 2005). Although they lack genes encoding S-layer proteins, L. gasseri encodes proteins annotated as ‘aggregation promoting factors’ (Apf), which share several features with the S-layer proteins of L. acidophilus, alluding to the possibility of shared functionalities (Ventura et al., 2002). Specifically, S-layer proteins of lactobacilli have a high predicted pI, low sulfur amino acid content, and high hydrophobic amino acid content, similarly observed in the Apf proteins of L. gasseri. The genetic structure of Apf proteins is conserved across multiple strains of L. gasseri, encoded as two tandem genes spaced by a short intergenic region, characteristic of the arrangement of S-layer genes in lactobacilli. The S-layer proteins of L. acidophilus NCFM are implicated in modulation of the immune system and adhesion to Caco-2 cells, but whether the Apf proteins in L. gasseri exhibit similar functionality has yet to be investigated (Konstantinov et al., 2008; Goh et al., 2009). In addition, L. gasseri ATCC 33323 contains 14 mucus-binding proteins, six of which exhibit a signal peptide and four of which are predicted to be covalently linked to the membrane via sortase A cleavage of the LPTXG motif (Azcarate-Peril et al., 2008). The proteins contain multiple mucus-binding domains that show homology with functional mucus-binding proteins in L. reuteri 1063 and L. acidophilus NCFM (Roos & Jonsson, 2002; Buck et al., 2005). Lactobacillus gasseri also contains a putative fibronectin-binding protein, which may mediate adhesion to the extracellular matrix of mammalian cells. The protein has homology to annotated fibronectin-binding proteins in L. johnsonii NCC 533 and L. acidophilus NCFM, although their roles in adhesion have not been confirmed. The adhesion of multiple L. gasseri strains to Caco-2 intestinal cells was evaluated and indicated strain-specific adherence capacity, but was generally less than L. acidophilus NCFM (Azcarate-Peril et al., 2008). In comparison to other strains of lactobacilli, L. gasseri ATCC 33323 exhibited slightly higher adhesion capacity to Caco-2, HT-29, and LS-174T cell lines, but less than that of L. reuteri strains. In vitro assays offer a practical means of evaluating relative adhesion properties as well as establishing the genetic and mechanistic bases underlying observed phenotypes, but must be corroborated by in vivo determination of relevance. The contribution of each of the individual mucus-binding and fibronectin-binding proteins to adhesion of L. gasseri ATCC 33323 has yet to be investigated, in vitro or in vivo.

The broad metabolic capabilities observed in L. gasseri ATCC 33323 may facilitate transient colonization of the upper GIT. However, while encoding 20 various phosphoenolpyruvate-dependent phosphotransferase systems (PTS) for sugar uptake and the capability of fermenting several monosaccharides, L. gasseri ATCC 33323 does not ferment five-carbon sugars and does not encode a β-galactosidase (Azcarate-Peril et al., 2008). Instead, it encodes two PTS and four putative phospho-β-galactosidases for the metabolism of lactose, although some strains of L. gasseri do not ferment the disaccharide (Hammes & Vogel, 1995). Interestingly, even though L. gasseri is a prevalent microorganism in early gut colonization and is commonly isolated from breast milk, ATCC 33323 is unable to ferment breast milk oligosaccharides or fructo-oligosaccharides, unlike other closely related lactobacilli (Ward et al., 2006; Rodrigues da Cunha et al., 2012).

Antimicrobial activity and bacteriocin production

Many LAB demonstrate antimicrobial activity toward a broad range of other bacteria by means of producing several antagonistic compounds, including organic acids, hydrogen peroxide, and bacteriocins (Fig. 2). Bacteriocins are classified as proteinaceous antimicrobial compounds that kill closely related microorganisms. In Gram-positive bacteria, a select few bacteriocins (e.g. nisin, pediocin, and lacticin) have a broad spectrum of activity against Gram-positive bacteria in general, including pathogens (Abee et al., 1995; Delves-Broughton et al., 1996; McAuliffe et al., 1998; Rodríguez et al., 2002). The considerable diversity of bacteriocins necessitates classification based on biochemical characteristics and categorizes bacteriocins as (I) lantibiotics and (II) heat-stable proteins not containing lanthionine residues, divided into multiple subgroups (Cotter et al., 2005). The acidophilus complex microorganisms are well documented as bacteriocin producers, and many of the bacteriocins exert activity specifically toward closely related species and some enteric pathogens.

Figure 2.

Potential mechanisms of antagonism by Lactobacillus gasseri through production of antimicrobial metabolites. (a) Depiction of circular bacteriocin synthesis and potential mode of action of gassericin A. Bacteriocins are ribosomally synthesized and often undergo post-translational processing such as hydrolysis of leader peptides. In class II bacteriocins, hydrolysis occurs at a conserved gly-gly motif that is absent in circular bacteriocins, which are predicted to have a different processing site. Secretion of these bacteriocins is achieved using a dedicated ABC transporter and circularization occurs enzymatically. The host cell synthesizes an immunity protein containing transmembrane domains that is predicted to localize cationic residues on the cell surface and sterically inhibit deposition of the bacteriocin on the host cell membrane, preventing permeabilization of the membrane of the producer cell. Circular bacteriocins are thought to dimerize and localize on the cell membrane of Gram-positive pathogens, where they undergo conformational changes and integrate hydrophobic α-helices into the cell membrane, resulting in pore formation and cell death by dissipation of the proton motive force (PMF), as well as efflux of potassium and amino acids. Activity of these bacteriocins is limited to Gram-positive bacteria due to the exclusion barrier of the outer membrane in Gram-negative microorganisms. (b) Two potential pathways were identified in the L. gasseri ATCC33323 genome for hydrogen peroxide production. Both pyruvate oxidase and lactate oxidase require oxygen and the metabolic end products of glycolysis for which they are named. Production of hydrogen peroxide by lactobacilli is hypothesized to be a nonspecific antimicrobial, but has been demonstrated to contribute to killing of pathogens in vitro with potential implications for urogenital health. Susceptibility of microorganisms varies largely among both catalase-positive and catalase-negative targets. In some cases, hydrogen peroxide alone is not sufficient for killing of target organisms, but cumulative effects of antimicrobials produced by lactobacilli augment its lethality. The mechanisms for cell death by hydrogen peroxide is largely facilitated by passive diffusion into the cell and reaction with Fe2+ by the Fenton reaction to produce highly reactive hydroxyl radicals, which cause DNA damage, denature proteins, and disrupt the cell membrane. (c) The antimicrobial activity of organic acids is largely dependent on their respective pKa and the pH of the environment, since passive diffusion into the cell is achieved only by the protonated acid. Once in the neutral pH of the cytoplasm, organic acids deprotonate and contribute to intracellular acidification. The cell must expend energy to drive proton efflux by ATPase to maintain a neutral intracellular pH.

Lactobacillus gasseri has been reported to produce a number of bacteriocins, with the most well characterized being gassericin A from L. gasseri LA39, isolated from infant feces (Toba et al., 1991). Gassericin A is a cyclic protein with 74% of its amino acids being hydrophobic (Kawai et al., 1998a, b). It is heat stable and resistant to degradation by proteases, due to its compact secondary structure consisting mainly of α-helices (Kawai et al., 1998a, b, 2000; Maqueda et al., 2008). The bacteriocin production and immunity genes are encoded by seven gaa genes in the 33 kb conjugative plasmid pLgLA39, including structural genes for putative transport and immunity (Fig. 3) (Ito et al., 2009). Several strains of L. gasseri were examined for the distribution of plasmids similar to pLgLA39, and while plasmids with related replication machinery were identified, none contained gaa genes (Ito et al., 2009). The ability of the pLgLA39 plasmid to conjugate between closely related strains was demonstrated and the plasmid conferred both bacteriocin synthesis and immunity in the transconjugant (Ito et al., 2009). The in vitro activity of L. gasseri LA39 was demonstrated against Gram-positive pathogens, namely Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus in a strain-specific manner, and the purified bacteriocin exhibited activity in situ (Itoh et al., 1995; Kawai et al., 1998a, b, 2000). Gassericin A has similarities with acidocin B from L. acidophilus M46 and reutericin 6 from L reuteri LA6, with sequence identities at 98% and 100%, respectively. The high sequence similarities of the conjugative plasmids pLgLA39 and pLrLA6 encoding the bacteriocins gassericin A and reutericin 6, respectively, indicate that the bacteriocin genes likely were disseminated by horizontal gene transfer. This is corroborated by the fact that microbial isolates producing gassericin A and reutericin 6 both originated from the same infant (Itoh et al., 1995). Reutericin 6 was observed to have a narrower spectrum which was thought to be due to the difference of one D-alanine amino acid, resulting in disparate secondary structure and function (Itoh et al., 1995; Kawai et al., 2004a, b), but this hypothesis was later disproved when the structures were found to be identical (Arakawa et al., 2010).

Figure 3.

Nucleotide and predicted amino acid sequences of acidocin A and gassericin A, synthesized by L. gasseri LF221 and L. gasseri LG39, respectively. Acidocin A and gassericin A exhibit characteristics of class II bacteriocins, namely resistance to heat and proteolysis.

Lactobacillus gasseri LF221, previously identified as L. acidophilus, encodes two chromosomally located bacteriocin sequences for acidocin A and acidocin B (Fig. 3) (Majhenic et al., 2004). Biochemical analysis indicated that acidocin A and B are class II bacteriocins being heat stable, hydrophobic, and devoid of lanthionine residues. The genetic structure of acidocin A indicates a putative operon containing three open reading frames encoding the structural genes, the putative immunity protein, and a putative secondary component for the active mature acidocin A. The open reading frames for acidocin B share moderate homology with the functional two-component bacteriocin lactacin F from L. johnsonii, indicating that acidocin A and acidocin B in L. gasseri LF221 are independent on a mechanistic basis, contributing to the broad spectrum of activity from LF221. Lactobacillus gasseri LF221 inhibited Listeria innocua, S. aureus, and several species of Clostridium, demonstrating the potential to antagonize pathogenic as well as spoilage microorganisms (Bogovic-Matijasić et al., 1998). Interestingly, gassericin T from L. gasseri SBT2055 has high similarity with acidocin B from L. gasseri LF221, with 100% sequence identity to the structural and immunity genes. However, their disparate spectrums of activity suggest post-translational modifications of the bacteriocin peptides that impact target sensitivity (Kawai et al., 1998a, b, 2000, 2000). Another bacteriocin characterized from L. gasseri is gassericin KT7 from an infant isolate L. gasseri KT7 (Zhu et al., 2000). Gassericin KT7 is a heat-stable protein susceptible to proteolysis that has demonstrated bactericidal activity against a number of Gram-positive foodborne pathogens, including B. cereus, L. monocyotogenes, Clostridium botulinum, Clostridium perfringens, and S. aureus, but not against Gram-negative bacteria. The activity of gassericin KT7 was stable across a broad pH range (2.5–9.0) and 59% of the amino acids are hydrophobic. Other than sharing some biochemical characteristics with class II bacteriocins, it remains unknown how this bacteriocin relates to others that have been classified previously on nucleotide or amino acid sequence alone. These studies highlight the potential of bacteria within the acidophilus complex to provide a source of novel bacteriocins with unique activity and applications.

The specific role of bacteriocins in probiotic-mediated antagonism of pathogens in vivo was demonstrated in a mouse model challenged with L. monocytogenes. The study elegantly showed that salivaricin was solely responsible for the protective effect conferred by L. salivarius UCC118 in preventing infection and mortality. A bacteriocin-negative mutant deficient in salivaricin production failed to confer protection (Corr et al., 2007). In light of these results, it remains essential to investigate the potential application of novel bacteriocins from L. gasseri in preventing infection from Gram-positive enteric pathogens in vivo. Bacteriocins isolated from L. gasseri may also be applied in food preservation, as their heat stability and extensive pH range may afford a suitable shelf life in products. Moreover, their broad-spectrum activity is conducive to antagonism of spoilage microorganisms and pathogens alike. Studies identifying novel bacteriocins from microorganisms within the acidophilus group are an indication of the inherent strain-specific disparity and phenotypic diversity of the group, which necessitate a thorough investigation of the activity of each bacteriocin.

Lactobacilli produce organic acids as end products of fermentative metabolism, which exhibit nonspecific inhibition of pathogens through intracellular acidification (Fig. 2) (Ray & Sandine, 1992). As an obligate homofermentor, L. gasseri metabolizes glucose to > 85% lactic acid via the Embden–Meyerhof–Parnas pathway (Hammes & Vogel, 1995). The inhibitory activity of lactic acid is dependent on environmental pH, as protonated acids are not excluded by the outer membrane, in contrast to charged dissociated salts. Once internalized, the neutral intracellular pH causes deprotonation of the acid, disrupting the proton motive force and necessitating ATP expenditure to drive efflux of protons via ATPase. Organic acids also disrupt the outer membrane by protonating its constituents, leading to permeabilization (Alakomi et al., 2000).

Hydrogen peroxide is also a nonspecific antimicrobial synthesized by lactobacilli, the activity of which is dependent on passive diffusion through the cell exterior (Fig. 2). Hydrogen peroxide interacts with iron in the Fenton reactions, leading to generation of highly reactive hydroxyl radicals. Hydroxyl radicals induce oxidation of thiols in proteins, oxidation of membrane lipids, and cleavage of DNA (Russell, 2003), but susceptibility of cells to hydrogen peroxide-induced oxidative stress is dependent on the activity of host antioxidant defense systems. Production of hydrogen peroxide constitutes a significant selection criterion for probiotic candidates and is highly variable among strains of L. gasseri (Atassi et al., 2006; Strus et al., 2006). Genomic analysis of L. gasseri ATCC 33323 reveals genes with sequence identity to pyruvate oxidase and lactate oxidase enzymes, providing a potential genotype for the observed production of hydrogen peroxide by this strain (Azcarate-Peril et al., 2008; Pridmore et al., 2008). Although hydrogen peroxide can be responsible for killing activity, nonspecific antimicrobials work in concert to augment the inhibitory activity of L. gasseri by inducing multiple forms of cellular stress (Atassi & Servin, 2010).

Evidence for persistence in the GIT

Survival and activity in the GIT are requisites for probiotic activity and accordingly, it is necessary to establish the viability of potential probiotic candidates empirically (Fuller, 1989). Genomic analysis of L. gasseri reveals niche-related genes that may contribute to survival during GI transit in vivo (Azcarate-Peril et al., 2008), which have been substantiated by studies designed to detect and quantify levels of L. gasseri following consumption and GIT passage (Pedrosa et al., 1995; Fujimura et al., 2006; Takahashi et al., 2006). In some cases, detection of L. gasseri postconsumption in humans was associated with a positive influence on certain health parameters of the host, indicating a potential role in maintenance of host homeostasis (Fujiwara et al., 2001).

In one study, individuals fed with L. gasseri OLL2716 had detectable PCR amplification of DNA specific to L. gasseri OLL2716 from the mucosal layer 1 h after consumption, indicating that this strain penetrates the human mucosa (Fujimura et al., 2006). In a separate investigation, two groups of elderly individuals, one healthy and one with atrophic gastritis were fed L. gasseri ADH twice daily and samples collected from the stomach, small intestine, and feces (Pedrosa et al., 1995). The feeding regimen consisted of 11-day cycles of nonviable then viable 1010 CFU administration followed by a washout period. In the healthy individuals, L. gasseri ADH was not isolated from the small intestine or stomach, but was isolated in these locations from individuals with atrophic gastritis. In both treatment groups, L. gasseri ADH was isolated from the feces within 12 h of consumption at levels of 107 CFU g−1. In another trial, L. gasseri SBT2055 was administered daily at levels of 109, 1010, and 1011 CFU for 7 days to individuals in treatment groups for each dosage (Fujiwara et al., 2001). Prior to, during, and following consumption, levels of the bacterium in the feces of each individual were recorded over time. The results indicated dose-dependent and host-dependent levels of detection in each of the treatment groups. Twelve of the 26 individuals receiving the low and middle dosage had detectable L. gasseri SBT2055, achieving maximum log values of 104 and 106 CFU g−1 in the feces, respectively. The high dosage group displayed a maximum of 107 CFU g−1 in the feces. Consumption of L. gasseri SBT2055 at the high dosage was associated with a decrease in the putrefactive marker p-cresol in the feces and a decrease in the resident Staphylococcus population of the microbiota. In a separate study, consumption of L. gasseri SBT0255 was accompanied by an increase in fecal Lactobacillus counts and detection of SBT0255 by PCR with specific primers, up to 2 weeks following administration (Takahashi et al., 2006). Taken together, these studies highlight the ability of L. gasseri to transiently colonize in the GIT of humans, although comparison among strains is impossible due to the different feeding regimens, experimental design, and detection methods.

Maintenance of gut homeostasis

Overall gut health is a complex spectrum of homeostasis, regulation, health, and morbidity, which varies greatly on an individual basis due to genetic and environmental factors. Probiotic microorganisms have been suggested to contribute to maintenance of gut homeostasis in a multifaceted manner. Certain probiotic bacteria have the capacity to decrease GI transit time, regulate host metabolism, and improve epithelial barrier properties (Sherman et al., 2009; O'Flaherty & Klaenhammer, 2010). One example of how gut microorganisms have been suggested to regulate host metabolism is through the production of short chain fatty acids (Arora & Sharma, 2011). Consumption of L. gasseri CECT5714 and L. coryniformis CECT5711 daily for 4 weeks was implicated in increasing butyrate concentration in the human intestine (Olivares et al., 2006ab). The difference between the yogurt control group and the probiotic treatment group occurred at 2 weeks into the treatment, but was not observed at 4 weeks, indicating that a time-dependent response was responsible for the increase in butyrate. The consistency and volume of feces as well as intestinal function were reported to significantly improve with the treatment. In a separate study, gut health parameters were compared between two groups, one that received conventional yogurt and one that received a probiotic yogurt containing L. gasseri CECT5714 and L. coryniformis CECT5711 (Lara-Villoslada et al., 2007ab). Consumption of the probiotic yogurt increased IgA secretion in the mucosal layer, was implicated in reducing fecal cytotoxicity, and decreased Salmonella choleraesuis adhesion to intestinal mucins in the feces. A randomized, placebo-controlled double-blind multicenter study involving 169 members examined the efficacy of using a proprietary combination of L. gasseri and Bifidobacterium longum (Omniflora©) in mitigating acute diarrhea (Margreiter et al., 2006). The combination treatment decreased the duration and severity of acute self-limiting diarrhea, suggesting that consumption of L. gasseri contributed to maintenance of gut homeostasis. Combined, these studies substantiate the potential probiotic roles of L. gasseri in improving specific parameters related to gut health.

Lactobacillus gasseri may have application in maintaining serum and kidney homeostasis through the metabolic capacity to degrade oxalate in the GIT (Fig. 4). Oxalate is a toxic component in certain foods that is not metabolized by host mechanisms, but rather by oxalate-degrading bacteria in the GIT. Excessive or chronic intake of oxalate can result in kidney failure or disruption in the metabolism of calcium. The genome of L. gasseri ATCC 33323 encodes enzymes involved in oxalate catabolism, specifically formyl-coenzyme A transferase (frc) and oxalyl-coenzyme A decarboxylase (oxc) (Azcarate-Peril et al., 2008). Expression analysis indicated that both frc and oxc were co-expressed in an operon during oxalate degradation, in vitro. While oxalate degradation varies by strain (Azcarate-Peril et al., 2008), the results illustrated that L. gasseri may be used to prophylactically or therapeutically lower oxalic acid toxicity or renal complications. Lactobacillus gasseri Gasser AM63 demonstrated the ability to persist and degrade oxalate in a continuous culture simulator of the human colon microbiota without changing the overall population (Lewanika et al., 2007).

Figure 4.

Putative pathway by which Lactobacillus gasseri ATCC 33323 degrades oxalate. Oxalate is imported into the cell via a formate-oxalate antiporter which may contribute to establishing a PMF in some oxalate degraders. Once internalized, oxalate is activated with Coenzyme A through the action of formyl transferase, resulting in the release of formate. Degradation of oxalate occurs via enzymatic activity of oxalate decarboxylase, of which the products are CO2 and formyl-CoA. In vitro expression of frc and oxc are pH-dependent, exhibiting a twofold increase in expression at pH 5.5.

Probiotic yogurt containing L. gasseri OLL2716 was demonstrated to decrease the ulcer index in mm2 of acute HCl-induced gastric ulcers in rats, whereas nonfermented milk was shown to have no effect at the same dose (Uchida & Kurakazu, 2004). The inhibitory effect of the probiotic yogurt on ulcer development was abrogated, but not abolished with pretreatment of the rats with indomethacin, indicating a possible role for prostaglandin E2. Prostaglandin E2 causes increased secretion of mucus and decreased secretion of gastric acid, which may contribute to abatement of ulcers. Prostaglandin E2 expression was upregulated in the treatment group, further suggesting the capacity of OLL2716 to promote healing of ulcers in this manner. Live L. gasseri OLL2716 were similarly shown to accelerate the healing of acetic acid-induced ulcers in rats (Uchida et al., 2010). Interestingly, both live and irradiated L. gasseri cells inhibited development of HCl-induced lesions and increased expression of prostaglandin E2. These studies highlight a potential role for L. gasseri in maintaining gastric homeostasis and promoting healing of gastric lesions.

Lactobacillus gasseri in vaginal health

The vagina is a complex ecosystem where the microbiota can be dominated by a relatively small number of Lactobacillus species, but the composition can vary largely depending on race, physiological state, and time (Zhou et al., 2010; Ravel et al., 2011; Zhang et al., 2012). The presence and relative ratios of specific lactobacilli have been investigated for their role in maintenance of vaginal homeostasis and in particular, their inverse association with bacterial vaginosis (BV) (Ravel et al., 2011; Jespers et al., 2012). Lactobacillus gasseri has been well documented as a commensal of the vaginal mucosa and exhibits a negative correlation with BV (Kiss et al., 2007; Tamrakar et al., 2007; Jespers et al., 2012). Moreover, it has been reported to be a dominant species of Lactobacillus in the vagina of healthy women (De Backer et al., 2007; Yan et al., 2009). The impact of L. gasseri on vaginal health indicates that it may confer colonization resistance against pathogens by direct inhibition through lactic acid, hydrogen peroxide, and bacteriocins (Strus et al., 2006; Martín & Suárez, 2010), or by displacing them through competitive adhesion (Fig. 5) (Boris et al., 1998; Atassi et al., 2006). Specifically, in vitro analysis has revealed significant production of these respective antimicrobial activities by vaginal isolates of L. gasseri and has examined their cooperative activity against Gardneralla vaginalis and Prevotella bivia (Atassi et al., 2006; Atassi & Servin, 2010). The in vitro antimicrobial activity and established role as a constituent of a healthy vaginal microbiota has led to preliminary investigation of L. gasseri for clinical application in maintaining vaginal homeostasis (Larsson et al., 2008; Ehrström et al., 2010; Strus et al., 2012).

Figure 5.

Activity of Lactobacillus gasseri in the GIT. This figure depicts attributes and activities of various strains of L. gasseri discussed throughout the review. The complete mechanisms by which probiotic microorganisms elicit health benefits remain to be fully elucidated, but potential routes for L. gasseri to influence the GIT are depicted. Probiotic microorganisms modulate the mucosal and systemic immune systems through the interaction of microbial surface components with PRRs on APCs. Cytokines secreted by the APCs and intestinal epithelial cells activate resident lymphocytes and macrophages in the lamina propria, enhancing the mucosal immune response and potentiating immunoglobulin A (IgA) synthesis. Furthermore, APCs migrate to mesenteric lymph nodes and induce T-cell differentiation through presentation of microbial antigens and secretion of cytokines. The capacity to modulate the immune system also contributes to the innate adjuvanticity of L. gasseri in the delivery of vaccines. Lactobacillus gasseri may also contribute to homeostasis through the production of compounds antagonistic toward pathogens, potentially inhibiting their activity in the GIT.

In one trial, women diagnosed with BV were treated conventionally with antibiotics and then received a supplemental treatment with capsules containing 108–10 CFU of several strains of Lactobacillus, including L. gasseri LN40 (Ehrström et al., 2010). Follow-up examination of diagnostic parameters and colonization were performed 2–3 days after administration, after menstruation and at a fixed time of 6 months. Despite the short intervention period, a nonsignificant increase in cure rate was observed, with L. gasseri LN40 exhibiting the highest incidence of colonization in women receiving the capsules throughout the course of the trial. Similarly, women diagnosed with BV and treated with clindamycin were subsequently administered capsules containing 108–9 CFU of both L. gasseri DSM 14869 and L. rhamnosus DSM 14870 (Larsson et al., 2008). The intervention ran for 10 days or until menstruation commenced and after each menstruation, the treatment was repeated during 10 days for three cycles. At the end of the trial, 64% of the women receiving lactobacilli were free from BV compared with 46% in the placebo group. In contrast to the other clinical trials, a randomized, double-blinded, placebo-controlled study was conducted to determine the effects of oral administration of a capsule containing lactobacilli on vaginal health parameters (Strus et al., 2012). The capsule contained a combination of L. gasseri 57C, Lactobacillus fermentum 57A, and L. plantarum 57B containing 108 CFU constituted by 50% L. gasseri and 25% of the other respective strains on a weight basis. The capsule was administered daily for 60 days while the level of vaginal lactobacilli and health parameters were monitored at several time points throughout the course of the study. Transient colonization of subjects in the intervention group by the strains administered was variable, but observed in both the GIT and the vaginal mucosa. The treatment was associated with a decrease in vaginal pH, improvement in diagnostic parameters for BV, and an increase in total lactobacilli in the vagina.

Preliminary trials evaluating the therapeutic administration of L. gasseri in combination with other lactobacilli suggest potential for clinical application in the treatment of BV. However, these results must be substantiated by larger randomized, double-blinded, placebo-controlled studies examining the same strains to merit changes in clinical practice.

Immune system regulation

There has been a recent increase in research activity surrounding the mechanisms of immunomodulation by probiotic microorganisms. This is justified given that the ability of probiotic microorganisms to regulate both systemic and mucosal immunity provides a unique opportunity to mediate afflictions related to both systems (Fig. 5). Probiotic microorganisms modulate the immune system through the interaction of microorganism-associated molecular patterns (MAMPs) with pattern recognition receptors (PRRs) on antigen-presenting cells (APCs). The major MAMPs of probiotic microorganisms governing the immune response are lipoteichoic acids, peptidoglycan, S-layer proteins, and nucleic acids (Bron et al., 2011), as they interact with PRRs including C-type lectin receptors, NOD-like receptors, and toll-like receptors (TLRs) (Lebeer et al., 2010). The resulting cytokine profiles elicited from APCs govern the proliferation and differentiation of effector T cells, resulting in both the ephemeral and perennial immune responses to the bacterium. Lactobacilli commonly induce T-cell polarization toward a T-helper (Th)-1 response by eliciting IFN-γ and IL-12 secretion by APCs during antigen presentation to CD4+ T cells (Mohamadzadeh et al., 2005; Mohamadzadeh & Klaenhammer, 2008). Lactobacillus gasseri ATCC 33323 has been well characterized in its ability to interact with specific TLRs on the macrophage cell line HEK-293, as well as the cytokine profiles elicited when co-incubated with myeloid differentiated dendritic cells (DCs) (Stoeker et al., 2011). Lactobacillus gasseri ATCC 33323 was reported to preferentially interact with a TLR-2/6 heterodimer, with less activation of TLR-2 alone. Interestingly, TLR-2 interacts with a diverse array of MAMPs, including lipoteichoic acids, glycolipids, and peptidoglycan, suggesting these components of L. gasseri may be active in immune signaling (Zähringer et al., 2008). The cytokine profile elicited from DCs when co-incubated with L. gasseri ATCC 33323 indicated a pro-inflammatory response to the bacteria, as increased expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) occurred. Similar results were obtained upon co-incubation of DCs with L. acidophilus NCFM, indicating that the immune responses to both microorganisms were largely dictated by conserved MAMPs of lactobacilli (Stoeker et al., 2011). Lactobacillus gasseri TMC0356, previously mis-identified as L. acidophilus TMC0356, was exposed to a murine macrophage cell line and the cytokines elicited were quantified by ELISA (Morita et al., 2002). Lactobacillus gasseri TMC0356 elicited high levels of IL-10, IL-6, IL-12, and TNF-α, in accordance with the observation of cytokines elicited from DCs when exposed to L. gasseri ATCC 33323 (Stoeker et al., 2011). In another study, heat-killed L. gasseri TMC0356 was demonstrated to increase the pulmonary mRNA expression of IFN-α, IFN-β and IL-2 in senescence-accelerated mice (Kawase et al., 2012). These cytokines may contribute to the proliferation and differentiation of natural killer (NK) cells, which was observed in the analysis of splenic NK cells from mice fed L. gasseri TMC0356. In a separate study, heat-killed L. gasseri OLL2809 cells had the capacity to alter T-cell differentiation by suppressing CD4+ T cells in a MyD88-dependent manner. Purified microbial RNA also suppressed CD4+ T cells, suggesting that nucleic acids of L. gasseri may play a role in immunomodulation (Yoshida et al., 2011).

The immune response of murine Peyer's patch (PP) cells to L. gasseri TMC0356 and L. rhamnosus GG was compared in vitro and in vivo (Harata et al., 2009). Interestingly, when co-incubated with cells from the PP in vitro, L. gasseri TMC0356 elicited higher levels of IL-6, IL-12, IFN-γ, and IgA than did L. rhamnosus GG. In contrast, the immune response of PP cells to the intragastrically administered probiotic strains indicated that L. rhamnosus GG elicited higher levels of IFN-γ, IL-6, and IgA. The disparate results between the in vitro and in vivo comparison of these strains may arise from the different capacity of each to adhere to the epithelium and interact with PP cells, as well as the inherent strain-specific expression of immunomodulatory MAMPs that may manifest in differential immune responses in vivo. In humans, a combination of L. gasseri CECT5714 and L. coryniformis CECT5711 was demonstrated to stimulate NK cells and increase IgA levels secreted, but this effect was not seen for L. gasseri ATCC 33323 or L. gasseri TMC0356 (Olivares et al., 2006ab; Stoeker et al., 2011). Mucosal IgA secretion is mediated by epithelial cytokines, being elicited by transforming growth factor (TGF)-β and potentiated by IL-6 (Fig. 5) (Goodrich & McGee, 1999). Taken together, these studies highlight the potential of both live and heat-killed L. gasseri to influence the host immune system. The differential experimental methodologies and strain-specific nature of immunomodulation make strain to strain comparison challenging. Nonetheless, disparate in vitro and in vivo results signify the necessity of thorough investigation of immunomodulation by probiotic microorganisms in vivo to confirm the validity and relevance of results obtained from cell culture experiments.

Prevention of allergic response

The factors involved in the development of allergic response are not well understood, but there is evidence suggesting that probiotic microorganisms may confer a protective effect against the immunological imbalances contributing to this condition (Kalliomäki et al., 2001, 2003). Notably, neonates genetically predisposed to hypersensitivity may fail to undergo environmentally driven Th-1 responses mediated by the microbiota or exposure to pathogens. The result of which is a predominant Th-2 response that promotes IgE expression, as well as the activation of eosinophils and mast cells active in the allergic response (Ozdemir, 2010). It is reported that allergic individuals may have abnormal GI microbiota compositions, notably some low in lactobacilli, albeit that any causal relationship in this association remains unclear (Björksten et al., 2001; Sjögren et al., 2009; Johansson et al., 2011). It is also thought that allergy is exacerbated by decreased or ineffective modulation of the immune system by regulatory T cells, which normally act to suppress Th-1 and Th-2 responses alike (Galli et al., 2008). Consequently, the mechanisms by which probiotics may prophylactically inhibit the development of allergy are multifaceted. The propensity of probiotic microorganisms to increase epithelial barrier integrity and potentiate the expression of IgA may decrease epithelial permeability to antigens (Isolauri et al., 2008). Moreover, the ability of probiotic microorganisms to modulate the immune system and skew toward a Th-1 cellular response holds potential for abrogating the development of allergic responses in hypersensitive individuals, since the expression of Th-1-associated cytokines counter-regulates Th-2 responses (Holvoet et al., 2013). Furthermore, probiotic microorganisms that induce expression of IL-10 and TGF-β have the potential to downregulate inflammation in established hypersensitivity (Niers et al., 2005). Recombinant DNA technologies offer advances in oral immunotherapy strategies by targeted expression of allergens in concert with intrinsic immunomodulatory surface components to induce tolerance or skew toward a Th-1 response (Wells & Mercenier, 2008). Recent studies have also confirmed the utility of employing established genetic tools to generate probiotic derivatives tailored for targeting specific immunological imbalances (Mohamadzadeh et al., 2011; Khazaie et al., 2012).

Lactobacillus gasseri has been evaluated in eliciting immunological changes both in allergen-sensitized models and hypersensitive individuals. In one study, peripheral blood mononuclear cells (PBMCs) were purified from allergic and healthy individuals, co-incubated with allergens, and the cytokine profiles analyzed. Preincubation of the PBMCs with L. gasseri PA16/8 diminished Th-2 response-associated cytokines IL-4 and IL-5, and increased the Th-1-associated IFN-γ (Ghadimi et al., 2008). Interestingly, the effects were greater in the PBMCs from healthy rather than allergic individuals, suggesting that the predisposed immunological state of humans may influence the degree to which probiotic microorganisms can modulate their responses. The ability of L. gasseri TMC0356 combined with L. rhamnosus GG to alleviate perennial allergic rhinitis was evaluated in ovalbumin-sensitized guinea pig and Norway rat models (Kawase et al., 2006, 2007). The administration of the probiotic combination abrogated the increase in vascular permeability caused by local inflammation and a nonstatistically significant decrease in serum IgE was observed in both models. In a separate study, individuals with high levels of serum IgE and perennial allergic rhinitis were administered milk fermented with L. gasseri TMC0356 daily for a duration of 4 weeks (Morita et al., 2006). Following the treatment, the individuals exhibited decreased total serum IgE levels and decreased antigen specific serum IgE compared with the baseline control. An increase in Th-1-associated PBMCs was also associated with the treatment. A double-blind randomized clinical trial compared the immunological response of allergic children to conventional yogurt with a probiotic yogurt containing L. gasseri CECT5714 and L. coryniformis CECT5711 (Martínez-Cañavate et al., 2009). The treatment group consumed 200 g of a probiotic yogurt with 106 CFU g−1 of each strain daily for 4 months and immunological parameters were observed. The probiotic intervention group exhibited a decrease in serum IgE and an increase in IgA when compared to the conventional yogurt group. Furthermore, the probiotic yogurt group experienced an increase in CD4+ CD25+ T-regulatory cells and NK cells, although basophils and eosinophils were unaffected.

Lactobacillus gasseri has been insufficiently studied in the avenue of preventing allergic diseases, but nevertheless shows potential in reducing Th-2- and IgE-related immunological responses, possibly through promotion and stimulation of the Th-1 associated cytokines and cell types. These studies further contribute to the basis of probiotic-mediated regulation of the mucosal and systemic immune systems, although effective application through clinical therapy requires considerable work to be implemented.

Inhibition of Helicobacter pylori

Helicobacter pylori is a common gastric pathogen with infection resulting in acute mucosal damage and the possible clinical manifestation of peptic ulcers, gastritis, and gastric cancer due to chronic infection. Current treatments include administration of antibiotics, which often result in the alleviation of symptoms, but not necessarily eradication of H. pylori in the host. Consequently, remission can occur and the subsequent infection of H. pylori can be related to the antibiotic resistance of the infectious strain. Lactobacillus gasseri OLL2716 demonstrated a direct inhibitory effect on the in vitro and in vivo growth and colonization of several strains of H. pylori, consisting of clarithromycin-sensitive and -resistant subtypes of the pathogen (Ushiyama et al., 2003). Interestingly, gnotobiotic mouse studies have further indicated the potential of lactobacilli to eradicate H. pylori in vivo, or to prevent infection entirely when colonized with lactobacilli (Kabir et al., 1997; Aiba et al., 1998). IL-8 has been implicated in playing a major role in the development of mucosal inflammation and injury in H. pylori infections as it is a chemotactic factor for neutrophils. Chronic expression of IL-8 due to recurrent H. pylori infection may exacerbate mucosal inflammation and tissue damage (Crabtree & Lindley, 1994). In this regard, co-incubation of L. gasseri OLL2716 with a MK45 cell line infected with H. pylori inhibited expression of IL-8 when compared with the control (Ushiyama et al., 2003). Further investigation of the ability of L. gasseri OLL2716 to inhibit IL-8 yielded similar results, but indicated that the probiotic strain neither inhibited adhesion of H. pylori to infected cells nor interfered with TNF-α-induced IL-8 expression (Tamura et al., 2006). This suggests that the decrease in H. pylori-induced IL-8 expression was mediated by a mechanism independent of adhesion of the pathogen. The study also reported that the decrease in IL-8 secretion observed in the cell culture line occurred similarly in humans infected with H. pylori.

The ability of L. gasseri OLL2716 to inhibit H. pylori infection was also observed in human clinical trials. A trial with 31 participants infected with H. pylori were fed yogurt containing L. gasseri OLL2716 at a level of 107 CFU g−1 daily for 16 weeks, and the urea breath test was performed as a measure of infection. The probiotic yogurt decreased markers for H. pylori infection 2 weeks after consumption, but not during the treatment. The probiotic yogurt treatment group exhibited decreased H. pylori in antral biopsies as well as the infection marker of serum pepsinogen levels (Sakamoto et al., 2001). Another study evaluated the efficacy of L. gasseri in both prevention and treatment of H. pylori infection in children (Boonyaritichaikij et al., 2009). Lactobacillus gasseri OLL2716 was administered to 82 asymptomatic H. pylori-infected children for 1 year, who were subsequently assayed for a H. pylori stool antigen to monitor infection. In the treatment group, 42% of the participants had remission 6 months after cessation of treatment, but 24 of the participants were free from infection after 1 year. The consumption of L. gasseri as a preventative measure was not associated with a decrease in the incidence of H. pylori infection. Data from in vitro assays and animal models suggest a potential role for mitigation and treatment of H. pylori infection by administration of L. gasseri OLL2716, but more clinical trials will be needed to substantiate its clinical relevance.

Alleviation of symptoms due to viral infection

The mechanisms by which probiotic microorganisms limit duration of viral infection and the associated symptoms is increasingly being elucidated by in vitro and in vivo animal studies. Conventional Balb/c mice were intranasally administered L. gasseri TMC0356 and challenged by H1N1 influenza infection. Mice receiving the Lactobacillus treatment experienced decreased morbidity from infection and upregulated expression of pulmonary cytokines, including IL-1β, TNF-α, IL-10, and monocyte chemotactic protein 1 (Harata et al., 2011). Similarly, Balb/c mice were prophylactically fed heat-killed L. gasseri TMC0356 and challenged with influenza virus H1N1 (Kawase et al., 2012). Pulmonary virus titers, activation of NK cells, and the expression of cytokines were recorded after sacrificing the animals. In the TMC0356 treatment group, weight loss associated with infection was abrogated and pulmonary virus titers were decreased when compared with the control group. The mucosal epithelium of the treatment group also retained higher barrier integrity when compared with the control. The pulmonary expressions of several cytokines were upregulated, including those associated with the activity of NK cells. Specifically, increases in expression of IFN-γ, TNF-α, and IL-12 were observed. Heat-killed TMC0356 conferred a similar protective effect against H1N1 as live cells (Kawase et al., 2010). Although this was the case, different cytokines were analyzed in each experiment. Therefore, it is difficult to note any differential immunological responses that might have occurred due to heat treatment of the cells. In addition, it is challenging to associate any particular cytokines with the protective phenotype.

Human clinical trials are needed to determine the clinical relevance of administering probiotic microorganisms as a preventative measure to limit viral infection. Notably, a combination of L. acidophilus NCFM and Bifidobacterium animalis ssp. lactis Bi-07 decreased severity and duration of acute flu-like symptoms in children when compared with the control group (Leyer et al., 2009). Similarly, L. gasseri has been reported to decrease the severity of viral infection symptoms in a human clinical trial, possibly due to similar mechanisms as the related L. acidophilus NCFM. Induction of viral defense genes in cell culture indicates a possible mechanism for the action of L. acidophilus NCFM in limiting viral infections in humans. Lactobacillus acidophilus NCFM was demonstrated to induce viral defense genes via TLR-2, causing increased expression of IFN-β, and subsequently inducing IL-12 and TLR-3 in bone marrow-derived DCs (Weiss et al., 2010). A combination of L. gasseri PA16/8, B. longum SP 07/3, and B. bifidum MF20/5 was administered in a multicenter randomized, placebo-controlled study during two sessions of the flu season (de Vrese et al., 2005). Symptom scores, duration of infection, as well as the cellular immune response and fecal counts were recorded. Although there was no difference in incidence of viral infection, the treatment group had reduced severity of bronchial, pharyngeal and nasal symptoms, as well as reduced duration of symptoms and days with fever. The cellular immune response in the treatment group showed a significantly higher amount of cytotoxic T cells and CD8+ T cells when compared with the control, while other immune parameters remained the same. The study indicates that L. gasseri has human clinical relevance in abrogating symptoms and the duration of upper respiratory viral infections, but additional trials are needed to further substantiate the protective effect.

Delivery of biotherapeutics

The generally recognized as safe (GRAS) status of certain probiotic microorganisms and the genetic tools available will likely accelerate the use of designed probiotic microorganisms for the targeted delivery of biotherapeutics (Wells, 2011). Specifically, due to the propensity of probiotic microorganisms to survive GI passage and adhere to intestinal epithelial cells, they are able to effectively deliver targeted recombinant biotherapeutics that benefit the host, either metabolically or immunologically. The use of probiotics for recombinant biotherapeutic strategies offers a broad range of potential applications with significant potential for administration of safe and effective treatments (Wells & Mercenier, 2008).

The inherent genetic systems available for transformation of L. gasseri ADH were employed in the recombinant production of CC chemokines, which was augmented by the use of the φ-ADH phage to transduce the gene and insert multiple copies into the host chromosome, achieving high expression (Damelin et al., 2010). The genetic tools available in L. gasseri have been demonstrated to facilitate recombinant expression of biotherapeutics, including introducing biosynthetic genes for folate synthesis, as well as the expression of manganese superoxide dismutase (Mn-SOD) (Bruno-Bárcena et al., 2004; Wegkamp et al., 2004). Recombinant Mn-SOD may contribute to GI homeostasis by neutralizing reactive oxygen species (ROS), which play an integral role in tissue damage associated with colitis. Expression of Mn-SOD in the GIT ameliorated the histological inflammatory scores in an IL-10 deficient murine colitis model, indicating potential for the therapeutic use of recombinant L. gasseri in abrogating colitis symptoms (Carroll et al., 2007).

Probiotic microorganisms with an inherent capacity to modulate the systemic and mucosal immune systems are positioned to mitigate and potentiate the host response to particular antigens in the delivery of mucosal vaccines (Stoeker et al., 2011). In a promising experiment, mice were prophylactically fed with L. gasseri ATCC 33323 expressing recombinant protective antigen for anthrax toxin fused to a DC-targeting peptide. Those mice challenged with B. anthracis exhibited a 100% survival rate, whereas the control mice did not survive (Mohamadzadeh et al., 2010). Oral delivery of the recombinant protective antigen expressed by L. gasseri increased the protective antigen-specific antibody in the mice and upregulated cytokines associated with T-cell proliferation and immunity, indicating strong potential for the innate adjuvanticity of this strain to potentiate targeted delivery of vaccines. The protective effect of the recombinant vaccine enhanced by inclusion of the DC targeting peptide, suggesting that antigen processing and presenting occurred via DC signaling (Fig. 5). DCs sample lumenal antigens by extending cellular processes through the epithelium without disrupting tight junctions, or by M cell- and epithelial cell-mediated antigen translocation (Rescigno et al., 2001; Jang et al., 2004). Antigen-primed DCs then migrate to the T-cell and B-cell areas in Peyer's patches or mesenteric lymph nodes to present antigens, stimulating migration of lymphocytes to the common mucosa (Shreedhar et al., 2003; Jang et al., 2006).

Conclusion

Lactobacillus gasseri is a widespread commensal bacterium that inhabits human mucosal niches and demonstrates potential probiotic applications by fulfilling many desirable probiotic attributes. Due to recent advances and the cost effectiveness of genome sequencing technology, candidate strains that are considered for probiotic or therapeutic applications should be comprehensively sequenced, due to the necessity of proper identification and evaluation of safety. Genome sequencing facilitates pan-genome species and strain comparison that identify niche-specific factors as well as conserved genotypes between lactobacilli. Genome sequences enable the use of genetic tools for functional analysis of key genotypes involved in the mechanisms of probiotic activity and expedite the utility of recombinant applications in strains of interest. Consequently, each putative probiotic strain of L. gasseri should be sequenced and thoroughly characterized since safety and clinical efficacy cannot be extrapolated between strains. Increasingly, in vivo gene expression analysis of probiotic cultures and host tissues will continue to be used to determine relevant genotypes and regulatory networks involved in eliciting health benefits, but has yet to be performed with regard to L. gasseri. Furthermore, novel cell surface proteins specific to L. gasseri and L. acidophilus B complex members, such as the Apf proteins and other cell surface factors should be characterized to determine their roles in adhesion, immunomodulation or other host–microorganism crosstalk. Comprehensive understanding of host–microorganism crosstalk opens up avenues for future probiotic applications through effective tailoring of probiotic microorganisms for amelioration of specific immunological conditions and physiological maladies. The empirical evidence suggesting probiotic application for L. gasseri must be substantiated with randomized, double-blind placebo-controlled human clinical trials to establish the efficacy of specific strain-related health benefits.

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Discovery of Lactobacillus gasseri as a novel species

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In the early 1960s, when I was a researcher at Institut Pasteur (Paris, France), I isolated a collection of Lactobacillus strains from human stools and identified them by classical phenotypical tests. I found many different species and not only ‘Döderlein's bacillus’ (Lactobacillus acidophilus) as was usually believed at that time. However, among the strains identified as L. acidophilus, the (G+C) content ranged from 32% to 50%. As nucleic acid technologies were not yet available, another methodology had to be developed to clarify this discrepancy.

After having translated the classical textbook ‘The Microbial World’ into French, I spent two years in the laboratory of two of the authors, Roger Y. Stanier and Michael Doudoroff, at the University of California, Berkeley. There, we decided to carry out a comparative analysis of isofunctional enzymes, namely the D- and L-lactate dehydrogenases (D- and L-LDHs) using a method that had been established earlier to study the subunits of animal LDHs, according to electrophoretic migration in starch gels. In all Lactobacillus strains tested (except one), well separated spots of D- and/or L-LDH were obtained in agreement with the type of lactic acid produced in cultures. Six LDHs were purified and used to obtain rabbit antisera. They were analyzed on Ouchterlony plates made of a thin layer of agarose gel in which distinct wells received either the antiserum or a crude extract containing the antigen to be tested. In the vicinity of an antiserum well, pair-wise comparisons of crude extracts produced either a continuous line of precipitate (indicating complete identity of the two LDHs tested) or a continuous line with a spur (indicating partial identity of two LDHs tested). In these electrophoretic and immunological tests, a large number of L. acidophilus strains from various origins fell into three separate groups, each having a distinct (G+C) content.

In the beginning of the 1970s, large DNA-DNA hybridization studies were performed in several laboratories. With respect to L. acidophilus species, it turned out that the three groups that I had characterized by LDH analysis actually correlated with five groups found by DNA-DNA hybridization; thus, the LDH analysis was less discriminatory. One of these groups was named L. gasseri (Lauer, E., and Kandler, O. 1980. Lactobacillus gasseri sp. nov., a new species of the subgenus Thermobacterium. Zbl Bakteriol Mikrobiol Hygiene I Abt Originale C 1: 75-78). Back at the Institut Pasteur, I was the director of the General Microbiology course for graduate students from 1971 until my retirement in 1995.

By Francis Gasser (*1928)

Acknowledgements

Research efforts in the NCSU Klaenhammer group are supported by the North Carolina Dairy Foundation, Danisco/Dupont Nutrition & Health, the Southeast Dairy Foods Research Center, and Dairy Management, Inc as administered by the Dairy Research Institute. The authors thank colleagues who contributed to this work through peer-review and discussions, Yong Jun Goh, Sarah O'Flaherty, Emma Call, and Brant Johnson.

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